Research Article

In vitro Kinetics Study of Cerastes Cerastes Phospholipase A₂Using Olive Leaf Extract: A Fluorescence Approach

Enas A Sadawe¹, Asia Mohamed¹, Nesren M Magel¹, Salah M Bensaber¹, Massaud Salem Maamar², Anton Hermann³ and Abdul M Gbaj¹*

¹Department of Medicinal Chemistry, Faculty of Pharmacy, University of Tripoli, Libya

²Zoology Department, Faculty of Science, Tripoli University, Libya

³Department of Biosciences, University of Salzburg, Salzburg, Austria

*Corresponding author: Abdul M Gbaj, Department of Medicinal Chemistry, Faculty of Pharmacy, University of Tripoli, Libya, Tel: +218913556785; Email: abdulgbaj1@hotmail.com

Citation: Sadawe EA, Mohamed A, Mage NM, Bensaber SM, Maamar MS, Hermann A, Abdul MG (2018) In vitro kinetics study of Cerastes cerastes Phospholipase A₂using olive leaf extract: A fluorescence approach. J Pharmacol Clin Trials 2018: 01-10.

Received: 29th November, 2018; Accepted: 18th December, 2018; Published: 19th December, 2018

Abstract

Studying snake venom kinetics is crucial for developing risk evaluation approaches and determining the best timing and dose of antivenom needed to neutralize all venom in snakebite patients. Polyphenolic compounds have shown to inhibit toxic effects induced by snake venom proteins. The interaction of polyphenols with Phospholipase A₂ of Cerastes cerastes snake venom was investigated by fluorescence spectroscopy. The decrease in the fluorescence versus time was conducted at room temperature in 0.01 M Tris, 0.1 M NaCl at pH 7.4. The decrease in fluorescence was following a pattern of zero-order kinetics rate in which the fluorescence is decreasing linearly with time. This study is expected to offer additional information about the interactions of PLA₂with natural product that might lead to therapeutic drug.

Keywords AOLE; fluorescence; PLA₂

Abbreviations AOLE: Aqueous Olive Leaf Extract; PLA₂: Phospholipase A₂

Introduction

Snake venom causes considerable mortality and morbidity typically needing hospitalisation and sometimes causing enduring disabilities, and in severe cases may lead to death [1,2]. Delayed admittance to suitable medical facilities, short of antivenom, and incomplete treatments are believed the major causes to the high mortality and morbidity [3,4]. Snake venoms contain a combination of biologically active polypeptides and proteins (encompass around ninety to ninety five percent of a venom load), beside with other non-protein constituents including lipids, amines, carbohydrates, in addition to the inorganic salts [5]. The proteins and polypeptides available in the snake venom could be enzymes such as L-amino acid oxidases (LAAO)], phospholipase A₂, (PLA₂), serine proteases (SVSP), and metalloproteases (SVMP), and the non-enzymatic substances such as kunitz peptides (KUN), disintegrins (DIS)] and three-finger toxins (3FTx) [5]. The snake envenomation treatment is mainly involves of taken of specific antivenoms for the snake species or type concerned, and helpful care. Snake antivenoms could be defined as antibodies that are obtained from the animals plasma such as sheep horses or [6]. The antibodies which are taken intravenously could be antibody fragments [F(ab’)₂ or Fab] or IgG immunoglobulin or that react and neutralise the free venom in the plasma of the patient [6]. The dosage and timing of the antivenom administration is still mainly experimental and usually based on in vitro studies in animals by neutralization of the venom [6]. The study of snake venom kinetics provides significant idea about the time course of the exposure to venom. The kinetic studies allow for an improved determination of an adequate timing and dose of antivenoms administration obtained from animals or herbs. Phospholipases A₂ are class of enzymes (PLA2s-E.C. 3.1.1.4) that can be found extracellular and intracellular. They are able to catalyze the hydrolysis of SN two acyl bonds of SN-three-phoapholipids. The Intracellular PLA₂s are frequently membrane related and are concerned in phospholipid metabolism nevertheless, extracellular PLA₂s are also available profusely in the venom of insects and snake [7]. PLA₂s are extremely stable protein owing to the existence of disulfide bonds that assist in many biochemical studies such as kinetic which is the purpose in this paper. In addition, this stability makes the enzyme easy for biochemical and structural characterization in addition to their pathological effects and other bioactivities [8,9]. Phospholipases A₂ are the major venom constituent of snakes classified to genus Bothrops and display a wide range of biological effects includes cardiotoxicity, antiplatelet activities, myotoxicity, anticoagulant, hemolysis, and neurotoxicity [10-12]. Phospholipases A₂ contain 121 amino acids residues and its molecular mass was calculated to be 13566.7 Da [13,14]. Tryptophan (Trp) which is present in phospholipase A₂ shows different fluorescence emission characteristics that are reliant on the polarity of the surrounding environment about the Trp side chain [15]. Aqueous olive leaves extract (AOLE) of Olea europaea L. (Oleaceae) contains large number of phenolic compounds which classified into five recognized groups: flavones (diosmetin-7-glucoside, luteolin-7-glucoside, apigenin-7-glucoside, diosmetin and luteolin); catechin (flavan-3-ols), flavonols (rutin); and substituted phenols (hydroxytyrosol, vanillin, tyrosol, caffeic acid and vanillic acid). The oleuropein is the richest phenolic compound in olive leaves, followed by luteolin-seven-glucosides, hydroxytyrosol, apigenin-7-glucosides and verbascoside [16-18]. The main aim of this paper is to study the kinetics properties of Phospholipases A₂ which could interact with AOLE polyphenolic components at the molecular level to understand phospholipase A₂ –inhibitor interactions.

Materials and Methods

All experiments were performed in Tris buffer which contained 0.01M Tris, 0.1M sodium chloride, at pH 7.4). Deionized water obtained by glass-distillation and all highly pure analytical reagents were used all over experiments. Jenway pH-meter model 3510 (Staffordshire, UK) was used to determine the pH values Millipore filters of 0.45 mm (Millipore, UK) were used for all buffer solutions.

Absorbance spectra

Absorbance spectra were determined using 6505-Jenway UV-visible spectrophotometer (London, UK). The cuvette used was quartz cells of one-centimeter path length. The UV-Vis absorbance spectra were recorded in the two hundreds to five hundreds nanometer range, and spectral bandwidth of three nanometer. For the final spectrum of each solution analyzed baseline subtraction of the buffer solution was performed. The protein content of venoms samples was determined by the spectrophotometric method of Markwell., et al [19]. Bovine serum albumin (BSA, Sigma) was used for standard assays.

Fluorescence spectra

Fluorescence emission and excitation spectra were determined using spectrofluorometer (Jasco FP-6200, Tokyo, Japan). The cuvette used was fluorescence 4-sided quartz cuvettes of one-centimeter path length. The shutter was used as automatic mode to minimize photo bleaching of the studied sample. The selected wavelengths were chosen for tryptophan and tyrosine residues to provide aggregate excitation. The kinetic spectrum was corrected for the fluorescence background of the buffer. The plot of fluorescence kinetic emission of snake venom (Cerastes cerastes, 24.6 μg/ml) vs time (sec) from 0- 900 seconds using excitation of λ₂₈₀ nm was performed in 0.01 M Tris, 0.1 M NaCl at pH 7.4.

Preparation of aqueous Olea europaea leave extracts

Leaves of Olea europaea (olive trees) were collected from the Tripoli Centre, Novellien zone, Tripoli, Libya during October 2018. The leaves (5g) were cleaned with distilled water and then dried with the aid of cool air for about 20 minutes at room temperature. Dried leaves were homogenized using a HO4A Edmund Buhler homogenizer (GmbH, UK) along with fifteen milliliter of distilled water. The obtained aqueous solution was then filtered through a 0.45 μm Millipore filter (GHD Acrodisc GF, UK).

Venoms

Snake (Cerastes cerastes) venom was extracted by manual stimulation and were obtained in liquid and semisolid forms, respectively, from the Zoology Department, Faculty of Science, University of Tripoli (Libya) and stored at -20ºC until use. Venoms were added to 2 ml of 0.01 M Tris, 0.1M NaCl at pH 7.4.

Results and Discussion

Olive leaves contain many powerful antioxidant polyphenolic compounds which are able to decrease the fluorescence due to a structural change in the 3D configuration of the phospholipase A₂ molecule, leading to revealing of the hidden tryptophan residues. It has been reported that low molecular phenolic compounds can affect both the tertiary and secondary structure of the proteins as established by FTIR and circular dichroism [20-22] and hence can led to a significant tryptophan quenching of the phospholipase A_2.In addition, tryptophan quenching by polyphenols could result by many process such as molecular rearrangements, excited-state reactions, complex ground state formation, energy transfer, and quenching due to the collision [21]. Fluorescence quenching could be differentiated by two main mechanisms; dynamic and static and both of them could lead to a decrease in the fluorescence [21]. Recently, we proved that the fluorescence intensity of the two amino acids intrinsic fluorophores species, tryptophan (Trp) and tyrosine (Tyr), decreased on the addition of the AOLE. In addition, the possible correlation of the decrease in the fluorescence intensity of phospholipase A₂ on addition of fifty microliter of AOLE to various molecular interactions processes [23].

The fluorescence of phospholipase A₂ was investigated and studied deeply by our group [23]. The obtained kinetic fluorescence spectrum in this paper showed that the fluorescence of the phospholipase A₂ was decreased on addition of 50 µl of AOLE [Figure 1]. The interaction between phospholipase A₂ and AOLE constituents was confirmed by a decrease in fluorescence intensity with increasing time. This significant fluorescence change proposes a strong association between phospholipase A₂ and the AOLE constituents. Figure 1 also showed that the fluorescence intensity of PLA₂decreased in the presence of 50 µl AOLE, indicating that the microenvironments of amino acid fluorophores were affected by presence of the polyphenols of the AOLE. In order to explicate the response behavior in fluorescence is related to the AOLE, a set of control experiments were performed using only buffer instead of AOLE. No change in the fluorescence intensity was seen indicting the changes is attributed to the AOLE constituents. The decrease in the fluorescence intensity may also indicate that the polyphenols act as a quencher and their accessibility is dissimilar depending on the regions of interaction with PLA₂ and this consistent with the Cotrim proposal [24]. The emission of tryptophan (W68) at 350 nm of the PLA₂ obtained in Figure 1 before addition of AOLE (zero time) demonstrates that this amino acids residues under particular situation is easier exposed to the buffer and this is consistent with the literature [24].

PLA₂is tetramer and was called as A, B, C and D. In the solution the 2 dimers formed from monomers of A and B are in the asymmetric unit display diverse conformation states owing to glycosylation by TTEG (tetraethylene glycol) and have active site similar region. Nevertheless, the second dimer formed by molecules C and D have a region which is empty from active site. In all monomers the microenvironments created by amino acids have 8 tyrosines, which spread on hydrophobic region in addition to the hydrophilic surface and just one tryptophan in between the interface of the dimmers [25]. Furthermore, upon addition of AOLE lead to possible slight conformational changes induced by the polyphenols and this affect the microenvironments of the tryptophan and tyrosine and a hence a decrease in the fluorescence seen. This assumption was confirmed by many published studies using fluorescence resonance energy transfer (FRET) and molecular docking [25-29]. The decrease in the fluorescence was following a zero order (with rate constant, k=-0.011 obituary fluorescence unit/sec) because the rate is independent of concentration of the substrate, and is equivalent to constant k. The development of product performs at a rate which is linear with time. The addition of extra substrate does not increase the rat

Figure 1: Fluorescence perturbation of snake venom by addition of AOLE. Plot of fluorescence emission of snake venom (Cerastes cerastes) (24.6 μg/ml) vs time (sec) from 0- 900 seconds using excitation of λ280 nm in 0.01 M Tris, 0.1 M NaCl at pH 7.4. Kinetic spectrum was corrected for small background fluorescence contributions from the buffer solution and was scaled to visualize the kinetic effect.

Similar kinetic study was performed by Tanweer et. al using Bee venom PLA₂ and they found that tryptophan and tyrosine residues have fluorescence due to the hydrophobic environment [30]. When the enzyme was mixed with an equivalent molar of oleoyl imidazolide at pH eight a piercingly decease in the fluorescence emission was noticed and then declined gradually, undergoing a little red shift. The time course of the slow phase matched to a half-life of 5-minutes which is in reasonable accord with the kinetics of activation [30]. The logic explanation of these results is that the extremely hydrophobic reagent like in our case polyphenols bind to the PLA₂ very rapidly, disturbing the surroundings of one of the tryptophan residues, and then experiences a comparatively sluggish reaction in which the oleoyl group can be transferred to an acceptor residue; this will lead to additional perturbation of the tryptophan environment and eventually lead to a decrease in the fluorescence. It has been reported by Martinez-Gonzalez et al. that the PLA2 inhibition percentage increased in a hyperbolic trend, as the concentration of the polyphenolic compounds increased and the IC50 values of many polyphenols were determined and this confirms the enzyme inhibition [31].

Conclusion

In this paper, the kinetic interaction of polyphenols of AOLE with PLA₂ has been studied using fluorescence spectroscopy. The decrease in the fluorescence intensity versus time could be related to the interaction between polyphenols of AOLE constituents. The decrease in the fluorescence was following a zero order pattern. This study of binding is of great significance to understand chemical-biological interactions for future drug design, biochemistry, and pharmacology studies. Additionally, this study is expected to provide more information about the interactions of PLA₂with natural product in vision of a function as a therapeutic drug.

References

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